Kirill A. Martemyanov - US grants

[unreadable] DESCRIPTION (provided by applicant): G protein signaling pathways in the retina are critically involved in reception and transduction of visual stimuli. The physiological operation of these pathways is dependent on the tight control of signal duration mediated by the Regulators of G protein signaling (RGS) proteins. Our long term goal is to elucidate the functional role of RGS protein in the retina signaling as a necessary prerequisite to understanding visual dysfunctions and therapeutic means of their treatment. The main focus of this proposal is on the R7 family of RGS proteins that are expressed in the retina where they control the rate of G protein inactivation during visual signal transmission. Research over the past several years have established the functional role of one R7 RGS member, RGS9, which utilizes a complex network of macromolecular interactions to shape the response of photoreceptors to light. [unreadable] [unreadable] The central HYPOTHESIS of this study is that the functional principles of RGS9 in photoreceptors also govern the function of other homologous R7 RGS proteins in retina neurons. We, therefore, suggest to utilize the wealth of methodological approaches and concepts developed in the studies of RGS9 in photoreceptors to gain insights into the organization and functional regulation of R7 RGS proteins through their macromolecular interactions. Specifically, our hypothesis will be addressed in the following SPECIFIC AIMS: [unreadable] [unreadable] 1. To elucidate the molecular mechanism of Gbeta5 action. We will perform detailed molecular and kinetic analysis to analyze how Gbeta5 regulates the activity of R7 RGS proteins. [unreadable] [unreadable] 2. To determine the functional significance of R7 RGS association with their membrane anchor, R7BP (R7 Binding Protein) using mouse models. Using mouse transgenic and knockout technology we will address the role of this newly discovered regulator of R7 RGS proteins in the retina. [unreadable] [unreadable] 3. To identify G proteins regulated by R7 RGS proteins in retina neurons. These studies should provide a better understanding of the regulation of signaling in the retina and generate insights into the molecular mechanisms of G protein signal disruption that lead to visual disorders and blindness. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Adjustments to cardiac output are made on a beat-to-beat basis in response to internal and external stimuli, and are mediated by the changing balance of input from the sympathetic and parasympathetic branches of the autonomic nervous system. While parasympathetic input normally tempers the pro-arrhythmic influence of sympathetic activation, too much parasympathetic influence can predispose to atrio-ventricular block or atrial fibrillation (a key risk factor for stroke). Conversely, too little parasympathetic influence is associated with heart failure, and is an independent predictor of increased morbidity and mortality in patients with coronary artery or congenital heart disease, and following myocardial infarction. Collectively, these observations suggest that altered parasympathetic regulation of the heart likely contributes to a broad spectrum of cardiac dysfunction, while simultaneously highlighting the inherent therapeutic promise of manipulations designed to predictably alter parasympathetic tone in the heart. The premise of this project is that the diagnostic and therapeutic potential associated with the parasympathetic regulation of the heart cannot be fully-realized without a clearer understanding of the signaling pathway(s) mediating its influence. A critical signaling pathway mediating the parasympathetic influence on the heart consists of the type 2 muscarinic acetylcholine receptor (M2R) and the G protein-gated potassium channel IKACh; the M2R-IKACh signaling pathway is a major determinant of cardiac rhythmicity, and dysregulation of this pathway has been implicated in both atrial and ventricular arrhythmias. Work in the initial project period established that the RGS6/Gß5 complex accounts for much of the RGS- dependent negative modulation of the M2R-IKACh signaling pathway, and that loss of this inhibitory influence yields enhanced parasympathetic signaling in the heart. The goals for the next project period are articulated in two Specific AIMs that are designed to: (1) Identify molecules and mechanisms underlying the RGS modulation of parasympathetic signaling, and (2) Develop targeted approaches to manipulate parasympathetic signaling in the heart. The proposed studies employ innovative and complementary approaches that leverage the unique strengths and reagents of two research labs, along with the expertise of an extended group of collaborators with overlapping research interests. Successful completion of the proposed work will yield a detailed understanding of the RGS-dependent modulation of parasympathetic signaling in the heart, along with tangible insights into the therapeutic potential associated with direct pharmacologic manipulation of the IKACh channel. Collectively, these efforts will result in a sophisticated understanding of the molecular and cellular mechanisms that shape the parasympathetic regulation of the heart, knowledge that can be translated into improved diagnostic and therapeutic approaches for arrhythmias.

DESCRIPTION (provided by applicant): Primary torsion dystonias (PTD) are a group of movement disorders characterized by twisting muscle contractures, with dystonia as the only clinical sign (except for tremor) and in the absence of neuronal degeneration or an acquired cause. There are multiple genetic causes, with overlapping phenotypes. We have now identified a series of mutations in GNAL, encoding G?olf, in patients with early onset torsion dystonia (EOTD) who do not harbor mutations in TOR1A or THAP1. G?olf is a G protein that couples striatal dopamine D1 (D1R) and adenosine A2a (A2AR) receptors to adenylyl cyclase V. Therefore, it is expressed in striatal output medium size spiny neurons and cholinergic interneurons. Abundant evidence supports dysfunction of the basal ganglia in dystonia, although other regions, e.g. cerebellum and cortex, are also involved. Within the basal ganglia, the focus has been on the dopamine D2 receptor (D2R) and striatal cholinergic interneurons. Other than mutations in the tyrosine hydroxylase biosynthetic pathway, GNAL is the first EOTD gene that directly points to the DA signal transduction system as the origin of pathophysiology, particularly to D1R. TorsinA is a AAA-ATPase protein and Thap1 is a transcription factor. Their specific functions, however, remain enigmatic, particularly as to how their mutations result in dystonia. Therefore, the connection between G?olf and the nigrostriatal dopamine system allows for directed, comparative assays of this system in mouse models of the three forms of EOTD. The rationale behind these studies is that dissecting the direct effects and compensatory maladaptations in neurotransmission, particularly dopaminergic and adenosinergic, Gnal heterozygote-null mice will offer clues to pathophysiology in DYT1 (TOR1A) and DYT6 (THAP1) EOTD as well. In Specific Aim 1, it will be determined whether mutations in EOTD genes TOR1A, THAP1 and GNAL result in similar altered DA neurotransmission in the striatum as evidenced by DA level and release, G protein activity, and cAMP production. In Specific Aim 2, baseline and pharmacologically induced behavior will be analyzed in the same genotypes. The molecular counterparts of the behaviors will be assayed via measures of induction of phosphorylation of ERK and DARPP-32, following D1R, D2R, and A2AR receptor agonists and antagonists. In Specific Aim 3, RNA-seq will be performed in the Gnal+/- mouse and THAP1-C54Y knockin mouse, and compared to those in the Tor1a GAG+/-mouse (via collaboration) to identify downstream targets, particularly in neurotransmitter pathways. Identification of a final common pathway in different forms of EOTD will aid in directing discovery of therapeutic targets for this currently incurable disorder.

Summary Opioid drugs are the most widely used analgesics in clinic, and are also some of the most widely abused substances. The adverse actions of these drugs, including peripheral side effects, dependence and tolerance, severely limit their utility as prescription analgesics for long term pain management. The µ-opioid receptor (MOR) is the primary target of the analgesic and rewarding effects of opioids. Thus, efforts aimed at developing safer and more effective opioid treatments will require a much deeper understanding of MOR signaling. Our long-term goal is to use unbiased forward genetics to dissect the molecular organization of the MOR signaling network using whole-animal behavioral responses to opioids as a phenotypic readout. Towards this goal, we developed a transgenic MOR model (tgMOR), in which mammalian MOR is expressed in the nervous system of the nematode C. elegans. We found that tgMOR animals gain the ability to respond to opioids, and exhibit all the cardinal behavioral hallmarks of opioid responses seen in higher organisms including acute depressant effects, desensitization and tolerance. We further demonstrated key known molecular players that control opioid responsiveness in mammals play conserved functions in tgMOR worms. Taking advantage of this model, we completed an unbiased, forward genetic screen for modifiers of behavioral opioid sensitivity, and isolated a large number of mutants with altered opioid responses. We have developed a pipeline for discovery, identification and validation of genes responsible for phenotypes using a combination of whole genome sequencing, mapping and targeted CRISPR/Cas9 gene editing. Using this approach, we uncovered several known and novel genes that regulate opioid responsiveness in worms, and confirmed their effects on MOR signaling using cell-based assays with cultured mammalian cells. Our findings suggest an elaborate, largely unknown, network of players exists to regulate MOR signaling. Thus, the main effort of this project focuses on identifying and characterizing these players by analyzing tgMOR mutants isolated from our unbiased, forward genetic screen. Our first aim will be to identify the genes responsible for 1) hypersensitivity, 2) hyposensitivity, and 3) impaired tolerance by pursuing a subsets of mutants from each phenotypic category. In the second aim, we will validate and perform mechanistic studies on identified, conserved regulators of MOR signaling using a comprehensive platform of cell-based assays that monitor various aspects of MOR signaling. The third aim will focus on exploring the pharmacogenomics by which MOR impacts behavior. To do so, we analyze interactions between genetic MOR variants found naturally in the human population, FDA-approved opioid drugs, and different genetic backgrounds using a humanized tgMOR C. elegans platform. It is anticipated that these studies will advance our understanding of how opioids act thereby paving the way to the development of safer opioid therapeutics.